Toxins derived from the bacterium Clostridium difficile are the principal cause of antibiotic-associated diarrhea. Although it is known that many of the clinical manifestations of this and other infectious diarrheal states are due to involvement of intestinal muscle, the pathophysiological basis of muscle cell dysfunction is unknown. We propose that the effects of C. difficile on intestinal smooth muscle result from: 1) The direct effects of toxin B on smooth muscle membrane electrophysiology and intracellular calcium metabolism, 2) The indirect effect of toxin A via inflammatory mediators released by activated leukocytes, and 3) The modulation of enteric nerve activity resulting in changes of neurotransmitter or peptide release. The overall goal of this proposal is to determine the role of each of these phenomenon in the motility response of the intestine to C. difficile, and to ascertain their underlying cellular mechanisms. First, since our preliminary data suggests that toxin B causes inhibition of calcium conductance in intestinal smooth muscle, we will evaluate the membrane electrophysiological response to toxin by studies of excitation- contraction coupling in tissue strips and membrane electrophysiology in isolated cells. The degree to which the effects of toxin B are a function of changes of cystosolic calcium will be studied by correlating changes of cell size and free cytosolic calcium in enzyme-dispersed muscle cells, and measurement of calcium release in saponin-permeabilized cells. Second, the effects of toxins on enteric nerves will be directly studied by measurements of intracellular membrane electrophysiology, synaptic potentials in response to extracellular fiber stimulation, and response to exogenous agonists in myenteric and submucosal nerves in situ. Third, inasmuch as we have shown that toxin A in vivo results in the release of eicosanoids and that in vitro leukotriene results in muscle membrane depolarization, we will specifically study the role of leukotrienes C4 and D4 in mediating changes of membrane excitability in isolated smooth muscle cells and enteric nerves. These studies are expected to increase our understanding of the cellular basis of the motility response to C. difficile, and to enhance understanding of the general mechanisms by which infectious agents act on gastrointestinal muscle.
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